Polyacrylamide gel electrophoresis (PAGE) is widely used in biotechnology laboratories for the processing of biological samples to separate the biomolecules present in the samples for identification, and in some cases to quantify the separated species. Protein mixtures, peptide mixtures, and mixtures of DNA, RNA, and fragments of DNA and RNA can all be separated on polyacrylamide gels. For protein mixtures, a particularly useful form of PAGE is SDS-PAGE where the detergent sodium dodecyl sulfate (SDS) is included in the sample. All proteins consist of linked amino acid residues and each protein folds naturally into a three-dimensional shape that is characteristic of, and distinctive for, each individual protein. Thus, both the amino acid sequence and the shape of the folded protein differ from one protein to the next. In the presence of SDS, however, proteins become denatured, i.e., they unfold and thereby lose their characteristic three-dimensional shapes. All denatured proteins therefore have the same shape and can be separated in SDS-PAGE on the sole basis of their molecular weight. Since proteins differ in the numbers and types of amino acids, different proteins generally have different molecular weights, and the range of molecular weights of common proteins is large.
Certain proteins are very close in molecular weight, however, which makes them difficult to separate in SDS-PAGE. To separate such proteins, or to optimize their resolution, SDS-PAGE is typically performed in a discontinuous gel, i.e., a composite gel that consists of a “stacking” or “stacker” gel cast above a “resolving” (also known as a “separating”) gel, where the stacker gel has a lower polyacrylamide concentration and thus a higher porosity. The stacker and resolving gels meet at an interface where the stepwise change in porosity occurs, and in certain cases the interface also includes a stepwise change in pH from approximately neutral or slightly acidic (pH 6.6 to 7.0) in the stacker gel to basic (pH 8.6 to 9.0) in the resolving gel. Despite these discontinuities, solutes, buffer solutions, and electric currents can travel freely across the interface from one part of the gel to the other. The combined gels are oriented in a vertical position with the stacker gel above the resolving gel, and samples are initially placed at the top edge of the stacker gel. An appropriately polarized electrical field is then imposed to cause the proteins in the samples to migrate downward through the stacker gel toward the interface and into the resolving gel. During the initial stages of the migration, the proteins collect at the interface in a single sharp band. The proteins then continue their migration and enter the resolving gel, where the single band separates into multiple bands representing individual proteins. The percentage of acrylamide in the resolving gel can be varied to achieve optimal separation of the proteins. In general, however, large proteins will separate most readily in resolving gels of relatively low percentage while small proteins will separate most readily in resolving gels of high percentage.
Unfortunately, casting a gel is a time-consuming and labor-intensive procedure, and for this reason it has become commonplace for researchers to purchase pre-formed gels rather than to cast a gel at its point of use. Purchased gels are typically referred to as “precast gels” while those prepared in the laboratory are referred to as “hand-cast gels.” Manufacturers of precast gels have developed gels that have a number of advantages over hand-cast gels. For example, the resolution in a polyacrylamide gel tends to decline after a few days of storage, and for this reason hand-cast gels are typically prepared shortly before use. Manufacturers have addressed this problem by formulating precast gels in ways that increase their longevity to a year or more. Another difficulty concerns the electric field strength at which a gel is run and the length of time involved in completing a run. Hand-cast gels are typically run at low field strength to avoid heating the gel during the run, since heating causes artifacts in the experiment, and the lower the field strength the longer the time required to achieve protein separation. Manufacturers have responded to this problem by formulating pre-cast gels that can withstand high field strengths without showing anomalies due to heating. Disclosures of gel formulations that address these problems are found in Rowell et al., U.S. Pat. No. 8,282,800 B2 (Oct. 9, 2012), and Petersen et al., United States Pre-Grant Publication No. US 2010/0187114 A1 (Jul. 29, 2012).
Once a gel is run, the scientist typically either stains the gel in order to visualize the proteins separated within the gel or transfers the separated protein bands in the gel to a membrane for further processing or identification. Staining the gel is typically achieved by applying chemical stains or dyes which are often hazardous chemicals. Staining also takes time. Manufacturers of pre-cast gels have addressed this problem by formulating gels that enable proteins to be visualized without the use of stains or dyes, thereby eliminating the use of hazardous chemicals and reducing the amount of time required to detect the proteins. For example, Bio-Rad Laboratories (Hercules, Calif.) sells Stain-Free™ gel systems. The process of transferring proteins to a membrane is known as a “western blot,” and involves performing the transfer in such a way that retains the separation of the individual proteins, and particularly the relative locations of the individual protein bands in the gel. Such a transfer allows the scientist to perform analyses that cannot be performed on a gel. Such analyses include immunoassays, for example, since the antibodies used in immunoassays are too large to enter the gel efficiently but can easily contact proteins on a membrane. All proteins, including those in stain-free gels, can be blotted to a membrane, adding further to the convenience of the scientist and the speed of the experiment. Some precast gels, like Bio-Rad Laboratories' TGX and TGX Stain-Free gels allow quicker transfer of proteins from gel to membrane.
While pre-cast gels have advantages, they also have disadvantages. One disadvantage is that they are more expensive than hand-cast gels, since the manufacturer will pass on to the user the cost of making, storing, and distributing the gels. A second disadvantage is that the user is limited to the types of gels that the manufacturers make available. With hand-cast gels, the user can customize the resolving gel by personally selecting monomer solutions of specific compositions to meet the special needs or interests of a particular experiment, but such flexibility is typically unavailable with precast gels. Despite its benefits, therefore, hand cast gels typically lack many of the desirable properties of manufactured pre-cast gels, including the ability to store gels for future use, run the gels quickly, visualize proteins without staining procedures, and quickly transfer the protein bands from the gels to membranes and confirm successful protein transfer on membrane without staining